1. Field of the Invention
The present invention relates to the use of fluorescent retrograde tracers to determine neuronal connectivity. More specifically, the present invention relates to the use of the compound 2-hydroxy-4,4'-diamidinostilbene isethionate for the retrograde axonal labeling of brain cells for determining their projections, axonal branching and chemical distribution patterns.
2. History of the Prior Art
Understanding the function of nervous tissue necessitates understanding its structure. One method which scientists and researchers utilize to understand the structure of the nervous system involves the injection of selected strains and dyes into specific areas of the brain. The pathways containing these indicators may be traced by observing the system under a fluorescence microscope. The nature of neural tissue has been studied by observing how nerve cells are connected to each other via long processes called axons. It is now known that certain substances, such as enzymes or dyes that are taken up by the axon terminals of cells and brought back to the cells by physiological cellular mechanisms, can be visualized in the cell body. For example, a substance can be injected into one area of tissue such as the brain, and by examining the tissue several days after that injection, the same substance can be observed in another area of the brain. Based on this observation, it is determined that transport has occurred and it can be concluded that one area of the brain connects to another area. Knowing those connections helps in understanding how the nervous system works.
One type of transport that has been studied involves injection of the enzyme horseradish peroxidase into terminal areas of neuronal axons in the brian, from which it is taken back in the axon to the parent neuronal cell bodies. This backwards transport is known as retrograde axonal transport. The procedure involves the use of the enzyme in a complex histochemical procedure involving reacting the tissue with certain chromogens, such as diamidino benzidine and tetramethyl benzidine, in the presence of the substrate hydrogen peroxide. The reaction produces a colored precipitate that can be visualized with a light microscope.
The next significant improvement came with the discovery that certain colored fluorescent compounds such as Dapi-Primuline, Evans Blue, and Propidium Iodine resulted in retrograde transport when injected into the brain. The transport was observable with a sensitive fluorescence microscope. With the colored dyes and a fluorescence microscope, the projections from one cell to different areas of the brain may be observed. That is, it may be determined if a cell gives rise to axons that go to one, two, or more separate areas in the brain. By injecting one dye into one part of the brain that contains an axon terminal, and another dye into a separate area, within a short period of time the brain sections may be cut and examined. If a cell contains the fluorescent dyes, it may be concluded that the cell connects to the brain areas into which the dyes were injected.
The foregoing techniques are limited by certain key problems. First, the dye taken up by the ends of the axons is often also taken up by fibers, known as fibers of passage, that happen to be passing through the area into which the dye is injected. In such a case, it is difficult to determine whether the process is retrogradely labeling cells that terminate in a given area, or is labeling cells because axons just jappened to be passing through the area going to another target area. Second, many of the dyed compounds are either toxic or are thought to be, or are, carcinogenic. Third, the compounds do not fluoresce brightly, or fade or degrade very rapidly. If the fluorophore (the fluorescent compound) fades rapidly, and before a picture can be taken, it becomes very difficult to get high quality photographs for analysis and publication. Fourth, most of the known compounds, after retrograde transport to the cell body, begin leaking out of the cells to nearby cells, resulting in a very confusing picture. Glial cells and neurons around the cells of interest are thus labeled, making it difficult to interpret the photographs. The problem is that the known dye compounds are not permanent or fast so as to remain within the cell. Fifth, the use of many of the particular dyes or fluorochromes is not compatible with other procedures because the dyes are washed out by solvents, their fluorescence is quenched by histochemical reagents, or they tend to fade too rapidly to be combined simultaneous with other procedures. Sixth, previously known fluorescent retrograde techniques have severe limitations because of the narrow ranges of permitted survival times of the animals after the injection. After injection, the dyes are brought back to the cell body at a specific unique rate. The time span between injection and analysis depends on the rate of transport, and is often very short. For example, the dye Nuclear Yellow requires a survival time of 18 hours after an injection of a 10 millileter axonal projection into the brain. If the survival time exceeds 24 hours, spurious results are obtained. If the survival time is less than 18 hours, no results are obtained because the dye does not reach the cell body in that time period. Presently known dyes have optimal survival time requirements within a very narrow range. In order to use two different colored dyes, either surgeries must be performed on different days, which jeopardizes the animal's life, or the dyes must be limited to those having the same exact survival time. A final limitation is that presently used retrograde tracers generally only label the nucleus or cytoplasm of a neuron, thus providing little information about the cytoarchitectronics (shape of the cell) of labeled cells.
A significant improvement in this technology occcurred when it was discovered that a stilbene derivative, 4, acetamido-4-isothiocyanostilbene2, 2'-disulphonic acid (SITS), could be used as a tracer, and was not taken up by fibers of passage (Schmued and Swanson, Brain Research 249 (1982) 137--141). The principal problem with the SITS compound was that it apparently included a mixed group of uncharacterized chemicals that varied from batch to batch and from manufacturer to manufacturer. Often, retrograde labeling was not obtained at all, depending on the SITS batch employed. Consistently reproducible results have not been obtainable with SITS.